The attachment of N-linked oligosaccharide chains to proteins is an important co-translational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. In recent years it has become clear that a group of human disorders, known collectively as the carbohydrate deficiency glycoprotein syndromes (CDGS), are the result of an impairment in glycoprotein synthesis. A key enzyme in the N-glycosylation process is oligosaccharyl transferase (OT). In yeast this enzyme, which is found on the endoplasmic reticulum, consists of nine different membrane protein subunits. The general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. The specific aims are to study in detail two of these subunits, Ostlp and Ost4p. Using a combination of biochemical and genetic techniques Ostlp has been shown to recognize -Asn-X-Thr/Ser- glycosylation sites. One objective will be to identify the domain in the primary structure of Ostlp that is involved in recognition of the glycosylation site sequence. Also, the lumenal domain of this membrane protein will be overexpressed to determine if it can recognize and bind to glycosylation sites in nascent chains and thereby inhibit their glycosylation by OT. By use of bifunctional crosslinkers the possible interaction of Ostlp with other subunits of OT will be studied. Another subunit of OT to be studied is Ost4p, which is a novel mini membrane protein of 36 amino acids. Site directed mutagenesis will be used to study how it interacts with the other subunits of OT. In particular it will be determined if it interacts with two other subunits of OT, Stt3p and Ost3p. The hypothesis to be tested by crosslinking, immunoprecipitation and suppressor mutation studies is that the interaction between these subunits occurs via their transmembrane domains. The possible existence of this mini membrane protein as a subunit of OT in mammals, including humans, will be studied.